Association between plasma CTRPs with cognitive impairment and neurodegeneration of Alzheimer's disease

Abstract Aims Recent evidence indicated the biological basis of complement 1q (C1q)/tumor necrosis factor (TNF)‐related protein (CTRP) 3, 4, and 14 for affecting brain structure and cognitive function. Thus, we aimed to investigate the association between plasma CTRPs with Alzheimer's disease (AD). Methods A multicenter, cross‐sectional study recruited patients with AD (n = 137) and cognitively normal (CN) controls (n = 140). After the data collection of demographic characteristics, lifestyle risk factors, and medical history, plasma levels of tau phosphorylated at threonine 217 (pT217), pT181, neurofilament light (NfL), CTRP3, 4, and 14 were examined and compared. Multivariate logistic regression analysis was applied to determine associations of plasma CTPRs with the presence of AD. The correlation analysis was used to explore correlations between plasma CTPRs with scores of Mini‐Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), Activities of Daily Living (ADL) scale, and Clinical Dementia Rating Sum of Boxes (CDR‐SB), and levels of plasma pT217, pT181, and NfL. Receiver‐operating characteristic (ROC) analysis and Delong's test were used to determine the diagnostic power of plasma CTPRs. Results Plasma levels of CTRP3, 4, and 14 were higher in AD group than those in CN group. After adjusting for conventional risk factors, CTRP3, CTRP4, and CTRP14 were associated with the presence of AD. In AD patients, CTRP3 was negatively correlated with scores of MMSE and MoCA, while positively correlated with ADL score, CDR‐SB score, pT217, and pT181; CTRP4 was positively correlated with CDR‐SB score, pT181, and NfL; CTRP14 was negatively correlated with MMSE score, while positively correlated with CDR‐SB score, pT217, and NfL. An independent addition of CTRP3 and 4 to the basic model combining age, sex, years of education, APOE4 status, BMI, TG, and HDL‐C led to a significant improvement in diagnostic power for AD, respectively. Conclusions All the findings preliminarily uncovered associations between plasma CTRPs and AD and suggested the potential of CTRPs as a blood‐derived biomarker for AD.


| INTRODUC TI ON
Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by progressive cognitive and social dysfunctions in clinical manifestations and extracellular plaques formed by βamyloid (Aβ) protein and intracellular neuronal fiber tangles formed by tau protein in representative pathological features. 1,2As the leading cause of dementia, AD accounts for approximately 60-80% of all dementia cases and has quickly become one of the most expensive, lethal, and worrisome healthcare burdens in this century. 3,4It is widely believed that AD is associated with multiple factors such as age, sex, genetics, and environment, but the exact pathogenesis has not been fully elucidated so far. 5eviously, we proposed the concept of the brain-visceral adipose tissue axis and preliminarily analyzed its potential influence on the development of AD, in which adipokines secreted by adipose tissue may play multiple regulatory roles in Aβ metabolism, tau phosphorylation, hippocampal neurogenesis, synaptic plasticity, and neuroinflammation. 6Of note, complement 1q (C1q)/ tumor necrosis factor (TNF)-related proteins (CTRPs) are a newly discovered adipokine family and are mainly secreted by the adipose tissue. 7CTRPs have a common characteristic structure including four different domains, one of which is homologous to adiponectin. 8To date, 15 members (CTRP1-15) have been subsequently termed and their relationships to various diseases have been uncovered; nevertheless, whether CTRPs are correlated with AD has not been reported.

Mounting evidence has proved complicated associations of
CTRPs with the brain.CTRP3 has the capacity of freely penetrating the blood-brain barrier (BBB) independent of glucose and lipid metabolism in vivo. 9The overexpression of CTRP3 attenuated neuronal apoptosis and neuroinflammation, and improved learning and memory abilities in elderly rats anesthetized with sevoflurane. 10petitive transcranial magnetic stimulation alleviated learning and memory impairment in rats receiving middle cerebral artery occlusion (MCAO) by activating the CTRP3-signaling pathway. 113][14][15] Lacking of CTRP4 impaired hippocampaldependent associative learning and memory abilities in mice in the fear-conditioning paradigm by altering expressions of cognitionrelated genes in hippocampus and cortex. 13CTRP14 generated by climbing fibers acts as a key anterograde signal-regulating synaptic formation and maintenance, 15 and is closely related to learning, memory, and synaptic conduction of mice. 16l the reports about CTRP3, 4, and 14 above suggest potential associations between CTRPs and a range of neurological diseases.
Given that cognitive impairment is the core symptom, and neuroinflammation, neuronal loss, and synaptic degeneration are pathological features of AD, we designed a retrospective study, established both AD and cognitively normal (CN) control groups, explored relationships of plasma CTRP3, 4, and 14 with the presence, main clinical characteristics, and current representative blood biomarkers of AD, and investigated their performances in the diagnosis of this disease.

| Study design and subject selection
All subjects were recruited from the outpatient departments of neurology of Sichuan Provincial People's Hospital, Chengdu Eighth People's Hospital, and Yunyang County People's Hospital from January 2022 to January 2023 (Figure S1).The diagnosis of AD was in accordance with the criteria for "Probable AD" of the National Institute of Neurological and Speech Disorders and Stroke/ Alzheimer's Disease and Related Disorders Association criteria (NINCDS-ADRDA). 17The subjects were excluded if they had: 18  points for more than 6 years of education, ≤20 points for less than 6 years of education, and ≤17 points for 0 year of education) and MoCA (≤26 points for more than 12 years of education and ≤25 points for <12 years of education). 18,19The Chinese version of the activities of daily living (ADL) scale which included a physical selfmaintenance scale and instrumental activities of daily living scale was used to evaluate the daily functional status of all subjects. 20,21s maximum score is 56 points with a higher score indicating the more impairment of daily living function.

| Statistical analyses
All statistical analyses were performed using IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA) and GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA).For continuous variables, the normality of the distribution was first determined by normality diagnostic graphs (histograms, Q-Q plots, and P-P plots) and Kolmogorov-Smirnov test.Then, they were expressed as mean ± standard deviation (SD) or median and interquartile range (IQR) according to results of the normality test.Furthermore, the differences between two groups were compared using t-test or Mann-Whitney U test depending on data distribution and homogeneity of variance.Categorical variables were expressed as proportions, and a chi-square test was applied in the comparison between groups.The influence of plasma CTRPs on the presence of AD was determined by multivariate logistic regression analysis, which allowed adjustment for the confounding factors.Clinical variables with p < 0.05 on univariate analysis and the general AD risk factors were incorporated into multivariate logistic regression analysis models.Results were expressed as odds ratio (OR) with corresponding 95% confidence interval (CI).Nominally correlations between plasma CTRPs with clinical characteristics and the representative blood-based biomarkers of AD were analyzed using Spearman's rank correlation or Pearson's correlation without the adjustment for multiplicity.The clinical value of adding CTRPs to the general risk factors for identifying or predicting the presence of AD was calculated with receiver operating characteristic (ROC) curves.Using DeLong's test, areas under the curve (AUC) between different models were compared.All tests were two-sided, and p < 0.05 was considered to be statistically significant.Goodness-of-fit of logistic regression models was assessed using the Hosmer-Lemeshow test.None of the above models displayed a Hosmer-Lemeshow chi-squared value yielding a p < 0.05, and therefore, none were rejected.

| Comparison of baseline data between two groups
A total of 277 subjects were recruited including CN group (n = 140) and AD group (n = 137), and the detailed enrollment process for subjects is shown in Figure S1.The demographic characteristics, BMI, lifestyle risk factors, medical history, cognitive function, daily living abilities, and blood tests of all subjects are shown in Table 1.

| Association between plasma CTRPs with the presence of AD
Using the univariate binary logistic regression analysis, the years of education, APOE4 status, BMI, TG, and HDL-C at baseline were Different combinations of CTRPs were also related to the presence of AD (Table 2).

| Correlations between plasma CTRPs with the clinical characteristics of AD
In

| Correlations between plasma CTRPs with representative blood biomarkers of AD
In the AD group, the plasma CTRP3 showed significant positive correlations with plasma pT217 (rs = 0.229, p = 0.007) and pT181   other correlations were not statistically significant in AD patients (Figure S6).Besides, most of the correlations between plasma CTRPs with pT217, pT181, and NfL in all subjects as well as sex-or APOE4-based subgroups showed statistical significance (Figure S7).

| Diagnostic power of plasma CTRPs for AD
To analyze the performance of plasma CTRPs and their combinations to distinguish AD patients from CN controls, a ROC analysis was conducted.Each indicator featured significantly high AUC, which DeLong's test was used to compare AUCs of these indicators mutually (Table S2).Both AUCs of CTRP3 (Z = 4.353, p < 0.0001) and CTRP4 (Z = 4.549, p < 0.0001) were higher than the AUC of CTRP14 (Table S2).
To determine whether CTRPs increased the diagnostic efficacy of conventional factors for AD, all aforementioned variables (age, sex, years of education, APOE4-positive, BMI, TG, and HDL-C) were used to establish the basic model for AD diagnosis, namely Model 0 where AUC of the ROC curve was greater than the random probability AUC (AUC = 0.825, p < 0.001).Then, CTRP3, 4, 14, and their combinations were added to Model 0, namely Models 1-7, respectively.
Furthermore, DeLong's test showed that except for Model 3, the diagnostic power of the other six models was higher than Model 0 (each p < 0.0001) (Table 3).
Finally, in order to demonstrate the model's generalization ability in the real world, an internal validation was conducted on 277 samples through bootstrap resampling for 1000 times.It showed that the model's validation efficiency and risk threshold were significantly higher than the random probability, and the calibration curve showed that the actual curve was close to the ideal curve reflected as the C-index (CTRP3: 89.3%, CTRP4: 98.8%, and CTRP14: 81.9%), indicating that the deviation between the models' predicted results and the actual results was small (CTRP3 CI: 95%, mean absolute error (MAE) = 0.019, CTRP4 CI: 95%, MAE = 0.012, CTRP14 CI: 95%, MAE = 0.021), suggesting that the model in this study had a good effect (Figure S9).

| DISCUSS ION
Previously, we proposed a new concept of the brain-visceral adipose tissue axis and sorted out its potential involvement in the pathogenesis of AD, in which a key mediator is adipokines produced by the adipose tissue. 6The adipose tissue is the main site producing and secreting CTRPs, and nominally traditional adipokines (e.g., leptin, resistin, adiponectin, chemerin, and lipocalin-2) have been widely studied on their associations with clinical characteristics and pathogenesis of AD. 6 In the present study, plasma levels of three CTRPs members were higher in the AD group than those in the CN group and were remarkably associated with the presence of AD, suggesting that CTRPs, as a newly discovered adipokine family, are also to be associated with AD.
Cognitive impairment and decreased abilities of daily living are core clinical characteristics of AD, so we employed MoCA and MMSE, ADL, and CDR-SB for the assessment of cognitive function, living status, and severity of dementia, respectively.In the present study, three CTRP members showed varying degrees of correlations with these four indicators of scores in the AD group, with CTRP3 performing more comprehensively.Likewise, we adopted pT181 and pT217 to evaluate the hyperphosphorylation of tau protein, and NfL to examine the degree of neurodegeneration.Each CTRP member showed correlations with a majority of these representative biomarkers in the AD group.It should be pointed out that both female sex and APOE4 represent the strongest risk factors for AD in addition to aging, even though there is currently no consensus on which one is stronger [25][26][27] ; thus, the similar correlations in the subgroups based on sex or ApoE4 status were further investigated following previous studies. 19,28All correlation analysis results indicate that CTRPs are associated with the symptomatic severity and neurodegeneration degree of AD.
As the molecular structural characteristic of the CTRPs family, the complement protein C1q represents the first subcomponent of the C1 complex which binds to various ligands and serves as a recognition target for several classical signal cascades. 29In detail, both CTRP3 and CTRP14 have four different domains including a signal peptide at the N-terminus, a collagenous domain with various lengths of Gly-X-Y repeats, a short variable region, and a globular C-terminal domain homologous to C1q, 30 while CTRP4 has two C-terminal globular domains connected by a short linker and lacks the collagen domain. 30CTRPs have multiple biological regulatory functions on immunity, metabolism, and inflammation, supporting their crucial role in a variety of diseases such as obesity, cardiovascular disease, and tumors. 13All the findings in the present study have preliminarily revealed the association between CTRPs and AD at the phenotypic level.It must be pointed out that although the diagnosis of "probable AD" under the NINCDS-ADRDA criteria is reliable, 31 multiple studies have confirmed that the criteria have approximately a sensitivity of 80% and a specificity of 70% using pathological-confirmed AD as a reference. 32To some extent, this limited the reliability of our further inferences about the involvement of CTRPs in the pathogenesis of AD from the findings of this study.Thus, we conducted the following literature review and theoretical analysis.
CTRP3 is permeable to the BBB not requiring the involvement of lipids and glucose in vivo, and then exerts biological effects through various signaling pathways. 9The overexpression of In divergent vertebrates such as humans, mice, and zebrafish, the expression of CTRP4 was predominantly detected in adipose tissue and central nervous system (CNS). 13,39In detail, CTRP4 is mainly expressed in spinal cord, midbrain, hindbrain, hypothalamus, cerebral cortex, and hippocampus in mice. 13Lacking of CTRP4 impaired associative learning and memory and changed the expression of activityregulated cytoskeleton-associated protein (Arc), phosphodiesterase 4D, and c-fos in the hippocampus and cortex in female mice. 13The overexpression of CTRP4 in the hypothalamus reduced TNFα and IL-6 levels in both brain and blood and inhibited hypothalamic NF-κB signaling and microglia activation in mice. 40During the ascending phase of herpes simplex encephalitis, the expression of CTRP4 in the brain was closely related to serum levels of IL-6 and TNFα, volumes of brain damage, and the decline in MMSE scores. 41In addition, CTRP4 was found to play a central role in energy metabolism. 12ernight fasting followed by feeding induced a hypothalamic expression of CTRP4. 12Intracerebral administration of CTRP4 inhibited food intake and altered overall energy balance by inhibiting the expression of hypothalamic appetite neuropeptides such as neuropeptide Y and agouti-related neuropeptide. 12CTRP14 is encoded by homologous genes highly expressed in both brain and adipose tissue and is believed to be closely related to cognitive and synaptic function. 42,43Lack of CTRP14 altered the ambulatory activity in a sex-and nutritional state-dependent manner.
The ambulatory activity was reduced in CTRP14 knock-out male mice in ad libitum fed, fasted, and refed states; however, female CTRP14

F I G U R E 1
Correlations between plasma CTRPs with cognitive function, daily living abilities, and severity of dementia in AD patients.Correlations between plasma CTRP3 with scores of MMSE (A), MoCA (B), ADL (C), and CDR-SB (D) in AD group.Associations between plasma CTRP3 with scores of MMSE, MoCA, and ADL in sex-based subgroup (E-G) and APOE4-positive-based subgroup (H-J).Relationships among plasma CTRP4 with CDR-SB score (K), plasma CTRP14 with MMSE score (L), and plasma CTRP14 with CDR-SB score (M).Relationships between plasma CTRP14 with MMSE score (N) and MoCA score (O) in male AD patients.The link between plasma CTRP14 with ADL score in APOE4-positive AD patients (P).AD, Alzheimer's disease; ADL, activities of daily living; APOE, apolipoprotein E; CDR-SB, Clinical Dementia Rating Sum of Boxes; CTRP, C1q/tumor necrosis factor-related protein; MMSE, Mini-Mental State Examination; MoCA, Montreal Cognitive Assessment.Correlation analyses were performed using Spearman's rank correlation or Pearson's correlation.p < 0.05 is considered to be statistically significant.far exceeded the random chance (AUC of 50%) (each p < 0.001, AUCs ranged from 0.682 to 0.945) with the cutoff values of CTRP3 (105.6011),CTRP4 (283.2391), and CTRP14 (16.9590) (Figure 3).

F I G U R E 3 F I G U R E 2
Abbreviations: APOE, apolipoprotein E; BMI, body mass index; CTRP, C1q/tumor necrosis factor-related protein; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride.